癌細胞對於鉑類藥物的抗藥性降低鉑類藥物抗腫瘤功效。因此，開發新的藥物或新的治療策略以克服抗藥性是迫切需要。在我們先前的研究中我們從人宮頸癌的SiHa細胞中建立了對奧沙利鉑具有抗藥性的亞系，將其命名為S3。在本研究中，我們的研究目的是1）探索由離子螯合劑與鉑類化療藥物組合產生一種新的治療方法，以及2）找出克服奧沙利鉑抗藥性的性的作用機轉。我們發現奧沙利鉑或是順鉑結合銅螯合劑D-青黴胺和鐵螯合劑Desferal在S3細胞有協同的毒殺作用。此外，D-青黴胺和Desferal顯著增加鉑和DNA結合，從而增加細胞對奧沙利鉑和順鉑的敏感性。分別的作用機轉上，D-青黴胺通過增加Sp1的轉錄，增加的銅吸收轉運蛋白hCtr1的表現。大量表現的Sp1誘導p53降解以致p53表現量減少。由於p53轉錄調控銅排除轉運蛋白ATP7A的表現，p53表現量減少也導致銅排除轉運蛋白ATP7A的表現減少。相同的，Desferal誘導Sp1的轉錄增加促進NF-κB的表現。值得注意的是，在S3細胞發現TfR1表現增加透過NF-κB轉錄調控。增加表現的Sp1促進了NF-κB的表達和NF-κB進入细胞核结合到TfR1啟動子區，增加TfR1的表現。在我們的動物研究，D-青黴胺和Desferal皆增加S3腫瘤對奧沙利鉑治療反應和顯著降低腫瘤體積，且不會造成實驗動物顯著體重減輕。綜合上述研究結果，我們對於降低奧沙利鉑的抗藥性提供了一種新的認識，離子螯合劑如D-青黴胺和Desferal與奧沙利鉑的組合顯著增加抗奧沙利鉑的腫瘤細胞的藥物的反應，有效地抑制了腫瘤的生長。

The development of platinum drugs resistance in cancer cells severely reduces its antitumor efficacy. Thus, a dire need exists to develop new drugs or novel therapeutic strategies to overcome drug resistance. We previously generated an oxaliplatin-resistant subline, named S3, from the human cervical carcinoma SiHa cells, and its resistance phenotype was well-characterized. In the present study, we aimed to 1) explore a novel therapeutic approach by combining ion chelators with platinum drugs, and to 2) decipher the underlying mechanisms for overcoming oxaliplatin resistance. We identified that copper chelator D-penicillamine and iron chelator Desferal exerted similar synergistic killing effects on S3 cells when combined with oxaliplatin and cisplatin. Moreover, D-penicillamine and Desferal significantly promoted platinum-DNA adducts formation and thus sensitized cells to oxaliplatin and cisplatin treatments. Mechanically, D-penicillamine and Desferal exerted two distinct routes to conquer the drug resistance. D-penicillamine promoted the expression of copper influx transporter hCtr1 by increasing Sp1 transcription. Sp1 overexpression induced p53 degradation and reduced the expression of copper efflux transporter ATP7A, which expression was regulated by p53. In the parallel study, Desferal induced the expression of Sp1, which promoted the expression of NF-κB. The overexpression of Sp1 increased the expression of NF-κB and translocated it into the nucleus to bind to the TfR1 promoter region, which subsequently increased the expression of TfR1. In our in vivo studies, both D-penicillamine and Desferal enhanced the response of S3 cells and tumors to oxaliplatin treatment and significant reduced the tumor volume without significant body weight loss. Taken together, this study provides a new insight that the combination of ion chelators such as D-penicillamine and Desferal with oxaliplatin significantly increases the drug response of oxaliplatin resistant cells and effectively suppresses tumor growth.

1. Boulikas T and Vougiouka M. Cisplatin and platinum drugs at the molecular level. (Review). Oncol Rep. 2003; 10:1663-1682.
2. Rosenberg B. Noble metal complexes in cancer chemotherapy. Adv Exp Med Biol. 1977; 91:129-150.
3. Hill JM and Speer RJ. Organo-platinum complexes as antitumor agents (review). Anticancer Res. 1982; 2:173-186.
4. Choy H, Park C and Yao M. Current status and future prospects for satraplatin, an oral platinum analogue. Clin Cancer Res. 2008; 14:1633-1638.
5. Gabbiani C, Magherini F, Modesti A and Messori L. Proteomic and metallomic strategies for understanding the mode of action of anticancer metallodrugs. Anticancer Agents Med Chem. 2010; 10:324-337.
6. Cepeda V, Fuertes MA, Castilla J, Alonso C, Quevedo C and Perez JM. Biochemical mechanisms of cisplatin cytotoxicity. Anticancer Agents Med Chem. 2007; 7:3-18.
7. Jansen BA, Brouwer J and Reedijk J. Glutathione induces cellular resistance against cationic dinuclear platinum anticancer drugs. J Inorg Biochem. 2002; 89:197-202.
8. Wang and Guo Z. The role of sulfur in platinum anticancer chemotherapy. Anticancer Agents Med Chem. 2007; 7:19-34.
9. Mu D, Tursun M, Duckett DR, Drummond JT, Modrich P and Sancar A. Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems. Mol Cell Biol. 1997; 17:760-769.
10. Howell SB, Safaei R, Larson CA and Sailor MJ. Copper transporters and the cellular pharmacology of the platinum-containing cancer drugs. Mol Pharmacol. 2010; 77:887-894.
11. Kuo MT, Chen HH, Song IS, Savaraj N and Ishikawa T. The roles of copper transporters in cisplatin resistance. Cancer Metastasis Rev. 2007; 26:71-83.
12. Klomp LW, Lin SJ, Yuan DS, Klausner RD, Culotta VC and Gitlin JD. Identification and functional expression of HAH1, a novel human gene involved in copper homeostasis. J Biol Chem. 1997; 272:9221-9226.
13. Samimi G, Katano K, Holzer AK, Safaei R and Howell SB. Modulation of the cellular pharmacology of cisplatin and its analogs by the copper exporters ATP7A and ATP7B. Mol Pharmacol. 2004; 66:25-32.
14. Chen CC, Chen LT, Tsou TC, Pan WY, Kuo CC, Liu JF, Yeh SC, Tsai FY, Hsieh HP and Chang JY. Combined modalities of resistance in an oxaliplatin-resistant human gastric cancer cell line with enhanced sensitivity to 5-fluorouracil. Br J Cancer. 2007; 97:334-344.
15. Kuo MT, Fu S, Savaraj N and Chen HH. Role of the human high-affinity copper transporter in copper homeostasis regulation and cisplatin sensitivity in cancer chemotherapy. Cancer Res. 2012; 72:4616-4621.
16. Chen SJ, Kuo CC, Pan HY, Tsou TC, Yeh SC and Chang JY. Mechanistic basis of a combination D-penicillamine and platinum drugs synergistically inhibits tumor growth in oxaliplatin-resistant human cervical cancer cells in vitro and in vivo. Biochem Pharmacol. 2015; 95:28-37.
17. Song IS, Chen HH, Aiba I, Hossain A, Liang ZD, Klomp LW and Kuo MT. Transcription factor Sp1 plays an important role in the regulation of copper homeostasis in mammalian cells. Mol Pharmacol. 2008; 74:705-713.
18. Katano K, Safaei R, Samimi G, Holzer A, Rochdi M and Howell SB. The copper export pump ATP7B modulates the cellular pharmacology of carboplatin in ovarian carcinoma cells. Mol Pharmacol. 2003; 64:466-473.
19. Safaei R and Howell SB. Copper transporters regulate the cellular pharmacology and sensitivity to Pt drugs. Crit Rev Oncol Hematol. 2005; 53:13-23.
20. Ponka P and Lok CN. The transferrin receptor: role in health and disease. Int J Biochem Cell Biol. 1999; 31:1111-1137.
21. Richardson DR and Ponka P. The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta. 1997; 1331:1-40.
22. Hoke EM, Maylock CA and Shacter E. Desferal inhibits breast tumor growth and does not interfere with the tumoricidal activity of doxorubicin. Free Radic Biol Med. 2005; 39:403-411.
23. Abeysinghe RD, Greene BT, Haynes R, Willingham MC, Turner J, Planalp RP, Brechbiel MW, Torti FM and Torti SV. p53-independent apoptosis mediated by tachpyridine, an anti-cancer iron chelator. Carcinogenesis. 2001; 22:1607-1614.
24. Liu Y, Cui Y, Shi M, Zhang Q, Wang Q and Chen X. Deferoxamine promotes MDA-MB-231 cell migration and invasion through increased ROS-dependent HIF-1alpha accumulation. Cell Physiol Biochem. 2014; 33:1036-1046.
25. Taetle R, Castagnola J and Mendelsohn J. Mechanisms of growth inhibition by anti-transferrin receptor monoclonal antibodies. Cancer Res. 1986; 46:1759-1763.
26. Brooks D, Taylor C, Dos Santos B, Linden H, Houghton A, Hecht TT, Kornfeld S and Taetle R. Phase Ia trial of murine immunoglobulin A antitransferrin receptor antibody 42/6. Clin Cancer Res. 1995; 1:1259-1265.
27. Van Reyk DM and Dean RT. The iron-selective chelator desferal can reduce chelated copper. Free Radic Res. 1996; 24:55-60.
28. Lui GY, Obeidy P, Ford SJ, Tselepis C, Sharp DM, Jansson PJ, Kalinowski DS, Kovacevic Z, Lovejoy DB and Richardson DR. The iron chelator, deferasirox, as a novel strategy for cancer treatment: oral activity against human lung tumor xenografts and molecular mechanism of action. Mol Pharmacol. 2013; 83:179-190.
29. Daniels TR, Delgado T, Helguera G and Penichet ML. The transferrin receptor part II: targeted delivery of therapeutic agents into cancer cells. Clin Immunol. 2006; 121:159-176.
30. Daniels TR, Bernabeu E, Rodriguez JA, Patel S, Kozman M, Chiappetta DA, Holler E, Ljubimova JY, Helguera G and Penichet ML. The transferrin receptor and the targeted delivery of therapeutic agents against cancer. Biochim Biophys Acta. 2012; 1820:291-317.
31. Elliott RL, Stjernholm R and Elliott MC. Preliminary evaluation of platinum transferrin (MPTC-63) as a potential nontoxic treatment for breast cancer. Cancer Detect Prev. 1988; 12:469-480.
32. Hoshino T, Misaki M, Yamamoto M, Shimizu H, Ogawa Y and Toguchi H. In vitro cytotoxicities and in vivo distribution of transferrin-platinum(II) complex. J Pharm Sci. 1995; 84:216-221.
33. Iinuma H, Maruyama K, Okinaga K, Sasaki K, Sekine T, Ishida O, Ogiwara N, Johkura K and Yonemura Y. Intracellular targeting therapy of cisplatin-encapsulated transferrin-polyethylene glycol liposome on peritoneal dissemination of gastric cancer. Int J Cancer. 2002; 99:130-137.
34. Kawabata H, Germain RS, Vuong PT, Nakamaki T, Said JW and Koeffler HP. Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo. J Biol Chem. 2000; 275:16618-16625.
35. Trinder D and Baker E. Transferrin receptor 2: a new molecule in iron metabolism. Int J Biochem Cell Biol. 2003; 35:292-296.
36. Kawabata H, Yang R, Hirama T, Vuong PT, Kawano S, Gombart AF and Koeffler HP. Molecular cloning of transferrin receptor 2. A new member of the transferrin receptor-like family. J Biol Chem. 1999; 274:20826-20832.
37. Daniels TR, Delgado T, Rodriguez JA, Helguera G and Penichet ML. The transferrin receptor part I: Biology and targeting with cytotoxic antibodies for the treatment of cancer. Clin Immunol. 2006; 121:144-158.
38. Hann HW, Stahlhut MW and Blumberg BS. Iron nutrition and tumor growth: decreased tumor growth in iron-deficient mice. Cancer Res. 1988; 48:4168-4170.
39. Torti SV and Torti FM. Iron and cancer: more ore to be mined. Nat Rev Cancer. 2013; 13:342-355.
40. Prost AC, Menegaux F, Langlois P, Vidal JM, Koulibaly M, Jost JL, Duron JJ, Chigot JP, Vayre P, Aurengo A, Legrand JC, Rosselin G and Gespach C. Differential transferrin receptor density in human colorectal cancer: A potential probe for diagnosis and therapy. Int J Oncol. 1998; 13:871-875.
41. Shinohara H, Fan D, Ozawa S, Yano S, Van Arsdell M, Viner JL, Beers R, Pastan I and Fidler IJ. Site-specific expression of transferrin receptor by human colon cancer cells directly correlates with eradication by antitransferrin recombinant immunotoxin. Int J Oncol. 2000; 17:643-651.
42. Gomme PT, McCann KB and Bertolini J. Transferrin: structure, function and potential therapeutic actions. Drug Discov Today. 2005; 10:267-273.
43. Head JF, Wang F and Elliott RL. Antineoplastic drugs that interfere with iron metabolism in cancer cells. Adv Enzyme Regul. 1997; 37:147-169.
44. Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010; 70:440-446.
45. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006; 58:621-681.
46. Chen SH, Kuo CC, Li CF, Cheung CH, Tsou TC, Chiang HC, Yang YN, Chang SL, Lin LC, Pan HY, Chang KY and Chang JY. O(6) -methylguanine DNA methyltransferase repairs platinum-DNA adducts following cisplatin treatment and predicts prognoses of nasopharyngeal carcinoma. Int J Cancer. 2015; 137:1291-1305.
47. Lin RK, Wu CY, Chang JW, Juan LJ, Hsu HS, Chen CY, Lu YY, Tang YA, Yang YC, Yang PC and Wang YC. Dysregulation of p53/Sp1 control leads to DNA methyltransferase-1 overexpression in lung cancer. Cancer Res. 2010; 70:5807-5817.
48. Perkins ND, Agranoff AB, Pascal E and Nabel GJ. An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation. Mol Cell Biol. 1994; 14:6570-6583.
49. Hirano F, Tanaka H, Hirano Y, Hiramoto M, Handa H, Makino I and Scheidereit C. Functional interference of Sp1 and NF-kappaB through the same DNA binding site. Mol Cell Biol. 1998; 18:1266-1274.
50. Tacchini L, Gammella E, De Ponti C, Recalcati S and Cairo G. Role of HIF-1 and NF-kappaB transcription factors in the modulation of transferrin receptor by inflammatory and anti-inflammatory signals. J Biol Chem. 2008; 283:20674-20686.
51. Fu S, Naing A, Fu C, Kuo MT and Kurzrock R. Overcoming platinum resistance through the use of a copper-lowering agent. Mol Cancer Ther. 2012; 11:1221-1225.
52. Morgan SL, Medina JE, Taylor MM and Dinulescu DM. Targeting platinum resistant disease in ovarian cancer. Curr Med Chem. 2014; 21:3009-3020.
53. Marini KD, Rossello FJ, Martelotto LG and Watkins DN. Using synthetic lethal screening to identify therapeutic targets for innately platinum resistant lung cancer.[abstract]. Cancer Research. 2014; 74 Suppl 19.
54. Woynarowski JM, Chapman WG, Napier C, Herzig MC and Juniewicz P. Sequence- and region-specificity of oxaliplatin adducts in naked and cellular DNA. Mol Pharmacol. 1998; 54:770-777.
55. Raymond E, Faivre S, Chaney S, Woynarowski J and Cvitkovic E. Cellular and molecular pharmacology of oxaliplatin. Mol Cancer Ther. 2002; 1:227-235.
56. Rixe O, Ortuzar W, Alvarez M, Parker R, Reed E, Paull K and Fojo T. Oxaliplatin, tetraplatin, cisplatin, and carboplatin: spectrum of activity in drug-resistant cell lines and in the cell lines of the National Cancer Institute's Anticancer Drug Screen panel. Biochem Pharmacol. 1996; 52:1855-1865.
57. Safaei R. Role of copper transporters in the uptake and efflux of platinum containing drugs. Cancer Lett. 2006; 234:34-39.
58. Cvitkovic E. Ongoing and unsaid on oxaliplatin: the hope. Br J Cancer. 1998; 77 Suppl 4:8-11.
59. Misset JL, Bleiberg H, Sutherland W, Bekradda M and Cvitkovic E. Oxaliplatin clinical activity: a review. Crit Rev Oncol Hematol. 2000; 35:75-93.
60. Gore ME, Fryatt I, Wiltshaw E, Dawson T, Robinson BA and Calvert AH. Cisplatin/carboplatin cross-resistance in ovarian cancer. Br J Cancer. 1989; 60:767-769.
61. Boven E, van der Vijgh WJ, Nauta MM, Schluper HM and Pinedo HM. Comparative activity and distribution studies of five platinum analogues in nude mice bearing human ovarian carcinoma xenografts. Cancer Res. 1985; 45:86-90.
62. Helleman J, Burger H, Hamelers IH, Boersma AW, de Kroon AI, Stoter G and Nooter K. Impaired cisplatin influx in an A2780 mutant cell line: evidence for a putative, cis-configuration-specific, platinum influx transporter. Cancer Biol Ther. 2006; 5:943-949.
63. Safi R, Nelson ER, Chitneni SK, Franz KJ, George DJ, Zalutsky MR and McDonnell DP. Copper signaling axis as a target for prostate cancer therapeutics. Cancer Res. 2014; 74:5819-5831.
64. Ishida S, McCormick F, Smith-McCune K and Hanahan D. Enhancing tumor-specific uptake of the anticancer drug cisplatin with a copper chelator. Cancer Cell. 2010; 17:574-583.
65. Holzer AK, Manorek GH and Howell SB. Contribution of the major copper influx transporter CTR1 to the cellular accumulation of cisplatin, carboplatin, and oxaliplatin. Mol Pharmacol. 2006; 70:1390-1394.
66. Lovejoy DB, Jansson PJ, Brunk UT, Wong J, Ponka P and Richardson DR. Antitumor activity of metal-chelating compound Dp44mT is mediated by formation of a redox-active copper complex that accumulates in lysosomes. Cancer Res. 2011; 71:5871-5880.
67. Cappellini MD. Exjade(R) (deferasirox, ICL670) in the treatment of chronic iron overload associated with blood transfusion. Ther Clin Risk Manag. 2007; 3:291-299.
68. Porter JB. Optimizing iron chelation strategies in beta-thalassaemia major. Blood Rev. 2009; 23 Suppl 1:S3-7.
69. Whitnall M, Howard J, Ponka P and Richardson DR. A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics. Proc Natl Acad Sci U S A. 2006; 103:14901-14906.
70. Chen CC, Chu CB, Liu KJ, Huang CY, Chang JY, Pan WY, Chen HH, Cheng YH, Lee KD, Chen MF, Kuo CC and Chen LT. Gene expression profiling for analysis acquired oxaliplatin resistant factors in human gastric carcinoma TSGH-S3 cells: the role of IL-6 signaling and Nrf2/AKR1C axis identification. Biochem Pharmacol. 2013; 86:872-887.
71. Matsunaga T, Hojo A, Yamane Y, Endo S, El-Kabbani O and Hara A. Pathophysiological roles of aldo-keto reductases (AKR1C1 and AKR1C3) in development of cisplatin resistance in human colon cancers. Chem Biol Interact. 2013; 202:234-242.